While strides have been made to reduce the lethality of septic shock, it is still a highly fatal condition that is on the rise. Immunomodulation may prove to be a viable treatment option for sepsis. Immunomodulation consists of the induction of an immunologically tolerant state typified by a reduced inflammatory response to infection. Tolerance induction by LPS has been shown to be highly effective in reducing both morbidity and mortality in murine sepsis models;however LPS is too toxic to be used in humans. Monophosphoryl Lipid A (MPLA) is an endotoxin derivative used as a vaccine adjuvant. When administered independently of other immuno-stimulants or antibiotics, MPLA elicits a tolerant phenotype characterized by an attenuated pro-inflammatory response to subsequent bacterial challenges;and also improved bacterial clearance and reduced mortality in murine sepsis models. The mechanisms contributing to this altered immunologic phenotype are not well understood. Preliminary results show an expansion of a mixed myeloid cell population (including immature myeloid suppressor cells) after MPLA priming, coupled with a relative decrease in lymphocyte numbers. Two Specific Aims are proposed to further assess the impact of MPLA-mediated immuno-modulation at the systemic and cellular levels. The goals of aim 1 are to characterize the effect of MPLA priming on the host response to infection by determining: 1. how long the altered immuno-phenotype persists after MPLA priming;2. the phenotype of leukocyte populations that are recruited by MPLA priming;and 3. the effect of MPLA priming on the pro-inflammatory and antimicrobial functions of specific leukocyte populations. Mice will be treated with MPLA prior to bacterial challenge. Specific leukocyte populations in the peritoneal cavity, and bone marrow will be characterized by flow cytometry. The functional importance of the identified leukocyte populations will be determined by antibody depletion, adoptive transfer, cytokine production, phagocytosis, oxidative metabolism, and bacterial clearance assays. MPLA may activate the TLR pathway in a MyD88-independent fashion, resulting in a predominant TRIF-based signaling cascade. Aim 2 will assess the ability to induce an immuno-modulated state in TLR4, MyD88, and Trif Knock out mice to determine the importance of these signaling pathways in vivo. This study is designed to characterize the molecular and cellular mechanisms involved in MPLA mediated tolerance induction. Greater mechanistic understanding is needed for MPL and other immunomoduolators to become clinically available treatment options. The use of such immune based therapies may help to reduce the dependence upon antibiotics, and stem the rise in antibiotic-resistant pathogens.